Cytochrome P450 electrochemical system

ABSTRACT

The present invention concerns class II cytochrome P450s, electrochemical systems for assaying cytochrome P450 catalytic activity, apparatus and methods for same.

The present invention concerns class II cytochrome P450s,electrochemical systems for assaying cytochrome P450 catalytic activity,apparatus and methods for same.

Cytochromes P450 constitute a large family of haem thiolate enzymes,found in bacteria, fungi, plants, insects and animals, which catalyse awide range of reactions, including hydroxylation of aliphatic & aromaticcarbons, epoxidation, oxidative deamination, sulphoxide formation,N-oxidation, N-hydroxylation, dehalogenation and N-, O- andS-dealkylation. These diverse reactions all arise from the fact thatcytochromes P450 are versatile oxygen-activation catalysts whichincorporate one atom of molecular oxygen into a broad range ofsubstrates with concomitant reduction of the other oxygen atom to water.These enzymes play important roles in the biosynthesis of secondarymetabolites in plants, in steroid metabolism in fungi and animals and,notably, in xenobiotic metabolism.

The considerable diversity of P450s results in man being able tometabolize a wide range of foreign chemicals. Metabolism by P450s has amajor influence on the pharmaco-toxicological properties of therapeuticdrugs, and consideration of this metabolism is a key part of the drugdesign process. Furthermore, several P450s are polymorphic in the humanpopulation, resulting in individual differences in drug and toxinmetabolism. In a different sphere, the ability of cytochromes P450 tohydroxylate unactivated carbons suggests that they have potentialapplications in synthetic chemistry.

In vivo, P450s require other proteins as electron donors. The class Ienzymes (most bacterial P450s, and the mitochondrialsteroid-metabolising enzymes) require an NADH-dependent reductase and aniron-sulphur protein, while the class II enzymes (e.g. the mammaliandrug-metabolising enzymes) simply require a flavoprotein NADPH-dependentreductase. The mammalian drug-metabolising mono-oxygenase system thusconsists of NADPH-cytochrome P450 reductase and a number of differentP450s, all of which are bound to the membranes of the endoplasmicreticulum. (By contrast, the only known bacterial class II P450, P450BM3 (CYP102) from Bacillus megaterium, is a soluble enzyme having bothP450 and NADPH-cytochrome P450 reductase activities in a singlepolypeptide chain.)

The assay of cytochromes P450, for example to study the metabolism of anew therapeutic agent, has classically involved incubation of thecandidate substrate with a microsomal membrane preparation from thecells of interest, together with NADPH as an electron source, followedby chromatographic analysis for product formation. Because severaldifferent P450s are expressed in most cell types, it is difficult inthis kind of assay to identify the specific P450(s) responsible formetabolism of the compound of interest.

More recently, therefore, individual recombinant P450s expressed inbacteria, yeast, insect or mammalian cells (see, for example Gonzalez,F. J. et al., 1995, Ann. Rev. Pharmacol. Toxicol., 36: 369-390;Pritchard, M. P. et al., 1997, Arch. Biochem. Biophys. 345: 342-354;Guengerich, F. P. et al., 1997, Curr. Opin. Biotechnol., 8: 623-628)have been used in combination with endogenous or recombinant P450reductase. These assays have generally also involved chromatographicanalysis; while NADPH consumption can be followedspectrophotometrically, the coupling between electron flow and productformation is variable and hence NADPH consumption may not be a reliableindicator of metabolism of a given compound.

It has recently been shown that electrons can be supplied to P450reductase electrochemically from a platinum electrode by means of amediator, cobalt sepulchrate (Estabrook, R. W. et al., Meth. Enzymol.,272: 44-51), and on the basis of this systems have been developed inwhich fusion proteins comprising NADPH-cytochrome P450 reductase and aclass II cytochrome P450 can be driven electrochemically (Estabrook, R.W. et al, 1996, Endocrine Res., 22: 665-671). In the case of the quitedifferent class I P450s, it has recently been shown that electrons canbe supplied electrochemically either via putidaredoxin (Reipa, V. etal., 1997, PNAS USA, 94: 13554-13558) or directly to the P450 if thelatter is immobilised in a lipid film (Zhang, Z. et al., J. Chem. Soc.Faraday Trans., 93; 1769-1774).

Further examples of prior art systems involving the use of Class I P450enzymes such as P450cam are given in Kazlauskaite, J. et al. (1996,Chem. Commun (Cambridge), 18: 2189-2190), Vilker, V. L. et al. (RedoxChem. Interfacial Behav. Biol. Mol., 1987, 105-112), Vilker, V. L. etal. (1997, Proc. Electrochem. Soc., Volume 97-6: 91-99) and GB 2312960.

The present invention overcomes the prior art disadvantages,particularly those associated with the use of a P450 reductase to supplyelectrons to a P450, and provides simple and convenient methods andarrangements for supplying electrons to class II cytochromes P450,particularly for assaying P450 catalytic activity.

Although it has not previously been possible to supply electrons to P450cytochromes without the use of P450 reductases, the present inventionobviates this need.

Thus according to the present invention there is provided a class IIcytochrome P450 attached to a graphite electrode. In contrast to theprior art this attachment does not require P450 reductase and thusprovides a substantial advantage over the prior art.

The P450 cytochrome is attached such that upon catalysis of a reactionby the enzyme, electron flow occurs from the electrode to the enzyme.

The P450 cytochrome may be attached to the electrode via dodecyldimethylammonium bromide (DDAB). It may be attached via otheramphiphilic molecules which are insoluble in aqueous solutions. Suchmolecules are well known and will be readily apparent to one skilled inthe art. They include Nafion (RTM) and phosphatidylcholine.

Also provided according to the present invention is apparatus fordetermining the catalytic activity of a class II P450 cytochrome,comprising a cell having a graphite electrode having attached to it thecytochrome P450, and also having electron flow detection means.

The inventors have also found that it is not necessary for the P450cytochrome to be bound to an electrode, and thus also provided accordingto the present invention is apparatus for detecting the catalyticactivity of a class II P450 cytochrome comprising a cell having amodified gold electrode contacting a solution containing the enzyme.

The gold electrode may be coated in 2,2-dithiodipyridine (Adrithiol) orby other polyfunctional molecules which can be adsorbed onto theelectrode and also interact specifically with the cytochrome P450. Thesemolecules will be readily apparent to one skilled in the art, andinclude those compounds disclosed as “mediators” by Christensen, P. A.and Hamnett, A. (“Techniques and Mechanisms in Electrochemistry”, 1994,Blackwell Academic Press, London, pp. 356-373) which is incorporatedherein by reference in its entireity. The polyfunctional molecules mayhave the structure of“Type IV” compounds discussed by Christensen andHamnett (supra) and Allen, P. M. et al., 1984, J. Electroanal. Chem.,178: 69 which is incorporated herein by reference in its entireity . Thecompounds may include 1,2 bis(4-pyridyl) ethylene, 4,4′ bipyridine, bis(4-pyridyl) bisulphide, or 4 mercaptopyridine. Such modified goldelectrodes are able to supply electrons to the cytochrome P450 yet donot need to be attached by strong chemical bonds to the cytochrome P450.

Compounds suitable to act as mediators of electron flow from andelectrode to an enzyme are also disclosed by R. W. Murray (Acc. Chem.Res., 1980, 13, 135) which is incorporated in its entireity herein byreference.

Generally speaking, cells may be three-electrode cells having a firstelectrode as described above (i.e. graphite or gold) and second andthird electrodes comprising a saturated calomel working electrode and aplatinum wire counter-electrode. Naturally, each electrode shouldcontact the solution to be tested for the presence of a substrate forthe P450 cytochrome.

The electron flow detection means may monitor current flow or maydetermine a steady state cyclic voltammogram, for example over the range−0.4V to +0.4V.

The cytochrome P450 catalytic activity may also result in the generationof peroxides, essentially a side-product when it is desired to assay thegeneration of a specific product resulting from catalysis. Thegeneration of peroxides results in electron flow and thus affects theresults obtained by the apparatus. Thus the apparatus may additionallycomprise means for detecting peroxide formation. Such means are wellknown and include colourimetric peroxide assay means and fluorimetricperoxide assay means. Alternatively, it may comprise an electrode whichdetects peroxide. Thus the electron flow required for the assayedperoxide generation can be subtracted from the total electron flow and amore accurate measure of product formation obtained.

A range of applications exists for the present invention, particularlyfor the screening of novel compounds as substrates to a cytochrome P450and for the use of P450 catalysis in synthetic chemistry. For example ahigh-throughput screening system for P450 substrates could be readilycreated. In the case of using P450s for synthetic chemistry, the abilityof P450s to catalyse hydroxylation of unactivated carbons, together withtheir broad substrate specificity, makes them attractive tools forsynthetic chemistry. However, the high cost of NADPH has to datehampered this. The ability of the present invention to drive reactionselectrochemically now makes their use more commercially attractive.

Thus the present invention also provides a method of determining thecatalytic activity of a class II cytichrime P450, comprising the use ofapparatus according to the present invention. It may be a method fordetermining whether a given compound is a substrate for a class IIcytochrome P450.

Also provided is a method of performing a synthetic chemical reaction,comprising the use of apparatus according to the present invention.

The invention will be further apparent from the following description,which shows, by way of example only, forms of electrochemical assays.

Experimental

Cytochromes P450 BM3 and P450 3A4 were expressed and purified asdescribed below. Electrochemical assays were performed using both enzymeimmobilised on a graphite electrode and also enzyme in solution with amodified gold electrode. The results showed that they were accurateassays for P450 catalytic activity.

Expression and Purification of Cytochromes P450

A number of different procedures have been described for thepurification of recombinant cytochromes P450 from different sources. Theprocedures used to obtain the proteins used in the present work aredescribed briefly by way of illustration; any other expression andpurification procedure which yields pure solubilised recombinant P450swould be equally satisfactory. The expression and purification of B.megaterium P450 BM3 has been published (Modi, S. et al., 1995,Biochemistry, 34: 8982-8988), and is therefore only summarised briefly;the purification of human P450 3A4 is not yet published and is thereforegiven in more detail.

B. megaterium cytochrome P450 BM3

The E. coli plasmid pJM20, encoding the expression system for the haem(P450) domain of cytochrome P450 BM3, described in (Miles, J. S. et al.,1992, Biochem. J., 288: 503-509), was obtained. The host strain used wasE. coli XL Blue 1 (supE44, hsdR17, recA1, endA1, gyrA46, thi, relA1,lac⁻F′ [proAB⁺lacI^(q) lacZDM15 Tn10 (tet^(r))]). The expression andpurification of the protein has previously been described. Thetransformed cells were grown in Terrific Broth medium containing 50μg/ml ampicillin and 50 μg/ml IPTG. The cells were harvested bycentrifugation at 10,000 rpm for 15 minutes and the resuspended cellsbroken by passage twice through a French Press (2.5 cm id×17 cm, PowerLaboratory Press, American Instrument Co. Inc.). Cell lysates werefractionated by ammonium sulphate precipitation, followed by ionexchange chromatography on a DEAE Sephacel (Pharmacia LKB) column, bychromatography on a hydroxyapatite column (Bio-Rad) and, finally, by gelfiltration on a Sephacryl S300 (Pharmacia LKB) column. This resulted inhomogenous active protein as shown by SDS-PAGE (with Coomassie bluestaining), by electrospray mass spectrometry, by the ratio of absorbanceat 418 nm and 280 nm (where a ratio of >1.7 is characteristic of pureprotein), and by the absence of any absorbance band at 420 nm in the COcomplex of the reduced enzyme. Protein concentrations were measured bythe method of Omura and Sato (1964, J. Biol. Chem., 239: 2379-2387)using a value of ε=77.5 mM⁻¹ cm⁻¹ at 418 nm.

Human Cytochrome P460 3A4

The E. coli plasmid pCW-3A4ompAhis6, encoding the expression system forhuman cytochrome P450 3A4, described by Pritchard, M. P. et al.,(1997,Arch. Biochem. Biophys., 345: 342-354), was obtained. The host strainused was E. Coli JM109 (Promega). 10-12 single colonies of E. coli JM109transformed with the plasmid pCW-3A4ompAhis6 were picked from a freshlystreaked plate and inoculated into 50 ml Modified Terrific Broth (perliter: 12 g, 24 g yeast extract, 2 g peptone, 4 ml glycerol, 17 mMpotassium di-hydrogen orthophosphate, 72 mM di-potassium hydrogenorthophosphate) supplemented with 1 mM thiamine, trace elements (6.1mg/l iron (III) citrate, 0.43 mg/l zinc chloride, 0.5 mg/l cobaltchloride, 0.5 mg/l disodium molybdate, 0.25 mg/l calcium chloride, 0.32mg/l copper chloride acidified with 0.13 mg/l boric acid and 25 μlconcentrated hydrochloric acid), 50 μg/ml ampicillin and 1 mMδ-aminolevulinic acid, in a 250 ml flask. Cultures were grown at 37° C.in a shaking incubator until reaching an OD at 600 nm of 0.6, at whichpoint isopropylthio-β-D-galactopyranoside (Gibco) was added to a finalconcentration of 1 mM, and the cultures were grown on for 20 hours in ashaking incubator at 30° C.

Cultures were transferred to 50 ml Fulcrum tubes and cooled on ice for15 minutes prior to pelleting the cells (20 minutes at 3000 rpm, 2800 g,Sorvall RT7 centrifuge). The cells were resuspended in 5 ml 2×TSE buffer(100 mM Tris-acetate, 500 mM Sucrose, 0.5 mM EDTA, pH 7.6), diluted with5 ml sterile water after resuspension. Lysozyme was added from a fresh10 mg/ml aqueous solution to a concentration of 0.25 mg/ml and the cellsswirled gently for 46 minutes at 4° C. The spheroplasts were spun down(20 minutes, 3000 rpm, Sorvall RT7 Centrifuge), the supernatantdiscarded and resuspended in 100 mM potassium phosphate, 6 mM magnesiumacetate, 20% v/v glycerol, 0.1 mM dithiothreitol, pH 7.6. Phenylmethylsulphonyl fluoride (PMSF) was added to 1 mM and the cells were lysed bysonication (SGE Soniprep, 4×20 sec bursts, full power). Cell debris wasspun down (20 minutes, 3000 rpm, Sorvall RT7 Centrifuge) and Emulgen 911was added (0.1% v/v final concentration, 10% v/v aqueous stock). Thedetergent/lysate solution was swirled at 4° C. for 45 minutes beforepelleting the insoluble material by ultracentrifugation (Sorvall UltraPro 80 ultracentrifuge, 60 minutes, 180,000 g, 4° C.).

A 1 ml Hi-Trap Agarose column was charged with 2 ml 100 mM nickelsulphate and washed with buffer A (20 mM potassium phosphate, 600 mMpotassium chloride, 20% v/v glycerol, pH 7.4). The solubilised lysatesolution was passed down the column which was then washed with 5 columnvolumes buffer A then 10 column volumes of buffer A containing 75 mMimidazole. The purified protein was eluted with buffer A containing 1 Mimidazole, the eluate being clearly identifiable by its intense redcolour. The protein solution was diluted 20-fold in buffer A anddialysed in prewashed EDTA-treated dialysis tubing against 2 L of bufferB (20 mM potassium phosphate, 0.2 mM DTT, 1 mM EDTA, 1 mM benzamidine,0.05% Emulgen 911, 60 mg/ml PMSF, pH 7.0) and loaded on to a Mono S(Pharmacia) column. The column was washed with 60 ml of buffer B and theP450 3A4 was eluted with a linear salt gradient (0 to 0.2 M KCl) inbuffer B. This resulted in homogenous active protein, as shown bySDS-PAGE (with Coomassie blue staining), by the ratio of absorbance at418 mn and 280 nm, and by the absence of any absorbance band at 420 nmin the CO complex of the reduced enzyme. Protein concentrations weremeasured by the method of Omura and Sato (supra).

Electrochemical Assay Systems

Two different methods have been developed for electrochemical assay ofP450s

Use of Enzyme Immobilised on a Graphite Electrode.

A graphite working electrode (0.2 cm² surface area) was prepared bypolishing first with 1 μm and then with 0.3 μm alumina powder. Asurfactant coating was then cast onto the graphite surface by applying10 μl of 0.1 M didodecyldimethylammonium bromide (DDAB) indichloroethane; the dichloroethane was evaporated gradually overnight.The DDAB-graphite electrode was then coated with protein by placing itin a solution of 30 μM cytochrome P450 in 0.1 M potassium phosphatebuffer, pH 8.0, for 2 hours.

A three-electrode cell was used, employing a saturated calomel workingelectrode, a platinum wire counter electrode and theenzyme-DDAB-graphite working electrode. The solution in the cell was 0.1M potassium phosphate buffer, pH 8.0. The potential was scanned betweenthe limits +0.4 to −0.4 V vs. SCE until a steady state cyclicvoltammogram was obtained. A blank current was obtained by scanning thepotential between the above limits at 0.1 V s⁻¹; three scans wereaveraged, and the current at the minimum of the cyclic voltammogram wasmeasured. The experiment was then repeated in the presence of substrate(typically 1 mM), and the difference between the current at the minimumof the cyclic voltammogram in the presence and absence of substrate wastaken as the assay reading.

Use of a Modified Gold Electrode

A gold electrode (0.031 cm² surface area) was modified by placing it ina solution of 0.1 M Adrithiol (2,2′-dithiodipyridine) in dichloroethaneovernight. The dichloroethane was allowed to evaporate by standing theelectrode in air for 10 minutes.

A three-electrode cell was used, employing a saturated calomel workingelectrode, a platinum wire counter electrode and the modified goldworking electrode. The solution in the cell was 0.1 M potassiumphosphate buffer, pH 8.0, containing, typically, 20 μM enzyme. Thepotential was scanned between the limits +0.4 to −0.4 V vs. SCE until asteady state cyclic voltammogram was obtained. A blank current wasobtained by scanning the potential between the above limits at 0.1 Vs⁻¹; three scans were averaged, and the current at the minimum of thecyclic voltammogram was measured. The experiment was then repeated inthe presence of substrate (typically 1 mM), and the difference betweenthe current at the minimum of the cyclic voltammogram in the presenceand absence of substrate was taken as the assay reading.

When it was desired to accumulate sufficient products for analysis, theexperiment was run as described, using either type of electrode, exceptthat the potential was maintained at a fixed value within the abovelimits for the necessary time period. The electrodes were then removedand the products extracted from the reaction mixture with ethyl acetate.After evaporation of the ethyl acetate the products were separated byHPLC and analysed by gas chromatography—mass spectrometry and/or nuclearmagnetic resonance spectroscopy. These analyses demonstrated that thesame products were obtained from the electrochemical assay as from theconventional assay employing NADPH and NADPH-cytochrome P450 reductase.

What is claimed is:
 1. A class II cytochrome P450 enzyme attached to agraphite electrode.
 2. A class II cytochrome P450 enzyme according toclaim 1, wherein said cytochrome P450 enzyme is attached such that uponcatalysis of a reaction by said enzyme, electron flow occurs from saidelectrode to said enzyme.
 3. A class II cytochrome P450 enzyme accordingto either one of claims 1 or 2, wherein said cytochrome P450 enzyme isattached to said electrode via didodecyldimethylammonium bromide (DDAB).4. A class II cytochrome P450 enzyme according to claim 1, wherein saidenzyme is not used in conjunction with P450 reductase.
 5. Apparatus fordetermining the catalytic activity of a class II cytochrome P450 enzyme,comprising a cell having a graphite electrode having attached to it saidclass II cytochrome P450 enzyme, and also having electron flow detectionmeans.
 6. Apparatus according to claim 5, wherein said cytochrome P450enzyme is attached to said graphite electrode via DDAB.
 7. Apparatus fordetecting the catalytic activity of a class II cytochrome P450 enzyme,comprising a cell having a modified gold electrode contacting a solutioncontaining said class II cytochrome P450 enzyme.
 8. Apparatus accordingto claim 7, wherein said gold electrode is coated with2,2-dithiodipyridine.
 9. Apparatus according to claim 5, wherein saidcell is a three-electrode cell, and said electrodes other than saidgraphite electrode are a saturated calomel working electrode and aplatinum wire counter-electrode.
 10. Apparatus according to claim 5 or7, wherein said electron flow detection means determines a steady statecyclic voltammogram.
 11. Apparatus according to claim 5 or 7,additionally comprising peroxide formation detection means.
 12. A methodof determining the catalytic activity of a class II cytochrome P450enzyme, comprising the use of apparatus according to claim 5 or
 7. 13. Amethod according to claim 12, wherein it is a method of determining thespecificity of said class II cytochrome P450 for a given substrate. 14.A method of carrying out a synthetic chemical reaction, comprising theuse of apparatus according to claim 5 or
 7. 15. Apparatus according toclaim 7, wherein said cell is a three-electrode cell, and saidelectrodes other than said modified gold electrode are a saturatedcalomel working electrode and a platinum wire counter-electrode.